阅读说明：本技术 一种生长细胞无油转化植物甾醇制备去氢表雄酮的方法 (Method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol by growing cells ) 是由 孟浩 刘喜荣 曾春玲 赵小娟 杨芳 于 2019-10-16 设计创作，主要内容包括：本发明涉及甾体类药物中间体的生产方法,具体来说是一种生长细胞无油转化植物甾醇制备去氢表雄酮的方法。发明内容包括3位羟基保护反应、生长细胞生物转化、水解、精制步骤。本发明以植物甾醇为原料生产去氢表雄酮,原料易得,降低了生产成本,收率更高；采取3位羟基保护的步骤在前,生长细胞生物发酵反应在后,直接加大了发酵底物在发酵液中的溶解度,有利于发酵的进行,反应路线更短,省掉了许多传统制备方法所需要的反应步骤和后处理步骤；从原料上减少了废油的产生,造成的污染小。(The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing dehydroepiandrosterone by converting phytosterol into growing cells without oil. The invention comprises the steps of protecting 3-site hydroxyl, biotransformation of growing cells, hydrolysis and refining. The invention takes the phytosterol as the raw material to produce the dehydroepiandrosterone, the raw material is easy to obtain, the production cost is reduced, and the yield is higher; the step of protecting the 3-position hydroxyl group is adopted, the solubility of a fermentation substrate in fermentation liquor is directly increased after the biological fermentation reaction of the growing cells, the fermentation is facilitated, the reaction route is shorter, and a plurality of reaction steps and post-treatment steps required by the traditional preparation method are omitted; the production of waste oil is reduced from the raw materials, and the pollution is small.)

1. A method for preparing dehydroepiandrosterone by oil-free transformation of phytosterol by growing cells is characterized by comprising the following steps:

(1) and (3) protecting hydroxyl: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;

(2) and (3) biological transformation of growing cells: carrying out slant culture and seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 strain, inoculating the strain into a transformation medium, and carrying out fermentation transformation on the phytosterol etherate to obtain a fermentation product;

(3) hydrolysis: extracting the fermentation product with ethyl acetate, heating under reduced pressure, concentrating, and hydrolyzing with hydrochloric acid to obtain hydrolysate;

(4) refining: and refining and purifying the hydrolysate to obtain a dehydroepiandrosterone product.

2. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol into phytosterol by using growing cells as claimed in claim 1, wherein the mass ratio of methylal to phytosterol in step (1) is 10-20: 1.

3. The method for preparing dehydroepiandrosterone by oil-free phytosterol conversion of plant sterols from growing cells according to claim 1, wherein said step (2) of seed culture of strain B-NRRL 3683 comprises the steps of:

(1) slant culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, agar 20g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;

(2) first-order seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;

(3) secondary seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 h.

4. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol into growth cells according to claim 1, wherein the conversion medium formula in step (2) comprises the following components in percentage by mass: 1-6% of corn steep liquor, 0.1-0.6% of sodium nitrate, 0.01-0.08% of diammonium hydrogen phosphate, 0.1-0.2% of PPE, 1-10% of phytosterol etherate, 0.1-40% of hydroxypropyl cyclodextrin and pH value of 7.5-8.0.

5. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol into plant sterol according to claim 1 or 4, wherein the phytosterol etherate is ground to 200 mesh.

6. The method for preparing dehydroepiandrosterone by oil-free transformation of phytosterol with growing cells according to claim 1, wherein the method for transforming growing cells in step (2) specifically comprises: preparing transformation medium according to the formula, sterilizing at 121 deg.C for 30min, cooling to room temperature, inoculating B-NRRL 3683 strain secondary seed liquid with mass percent of 10-20% under aseptic condition, and transforming at 28-32 deg.C, air flow of 0.5-1.0vvm, and tank pressure of 0.05-0.06 MPa.

7. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol from growing cells according to claim 1, wherein the mass fraction of hydrochloric acid in the step (3) is 5%, the material ratio is 1-20 parts by volume of ethyl acetate and 1.5-30 parts by volume of 5% hydrochloric acid; reducing pressure and heating to-0.08 MPa, 45 deg.C, and hydrolyzing for 5 hr, adding hydrochloric acid, heating to 60 deg.C, and hydrolyzing for 1-2 hr.

8. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol into growing cells according to claim 1, wherein the refining method in the step (4) specifically comprises the following steps: drying the hydrolysate at 70 ℃, dissolving by using methanol, carrying out suction filtration, carrying out reduced pressure concentration on the filtrate to a small volume, cooling to about 4 ℃, carrying out suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, pulping at 70 ℃ under reflux for 2-3h, cooling to 25 ℃, filtering to obtain a white-like solid, drying, adding methanol, heating to 70 ℃ for dissolution, concentrating under reduced pressure to paste, slowly cooling to 0-4 ℃, growing crystals for 2 hours, carrying out suction filtration, and drying.

9. The method for preparing dehydroepiandrosterone by oil-free conversion of phytosterol with growing cells according to claim 1, wherein the refining process of step (4) comprises the following steps of adding 3-6 parts by volume of methanol, 0.5-1 part by volume of petroleum ether, 2 times of methanol, 1-2 parts by volume of methanol for the first time, 2-4 parts by volume of methanol for the second time, reducing pressure and raising temperature at-0.08 MPa for 45 ℃ for 8 hours, and drying at 60 ℃ after crystal growth.

Technical Field

The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing dehydroepiandrosterone by converting phytosterol into growing cells without oil.

Background

Dehydroepiandrosterone (dehydroepiandrosterone)rone, referred to as DHEA for short), the chemical name is 3 beta-hydroxyandrost-5-en-17-one, and the molecular formula is C₁₉H₂₈O₂Is a kind of C₁₉The adrenal steroid compound is mainly secreted by adrenal cortex, and the gonad such as testis and ovary also have a small quantity of secretion, and its physiological activity action research range relates to resisting cancer, delaying senility, regulating immunity, resisting diabetes and preventing and curing tumor, etc. In addition, DHEA has a very important position in the synthesis of steroid drugs, and DHEA is used as an initial compound, and a plurality of drugs with important physiological activity and medical value can be obtained by modifying a parent nucleus or a side chain of the DHEA.

At present, the preparation method of dehydroepiandrosterone mainly comprises a chemical method and a microbial conversion method. The chemical method is mainly completed by means of multi-step chemical reactions, the traditional preparation process takes androstenedione as a raw material, and selectively reduces 3-position ketone group to beta hydroxyl group by a chemical method, but the product obtained by the chemical synthesis method often has a certain amount of 3-position alpha hydroxyl isomer and other impurities, the yield is low, the steps are complicated, and the pollution is easily caused by using various organic reagents. Patent No. CN201210316197.0 discloses a method for preparing dehydroepiandrosterone by microbial fermentation, which uses phytosterol as raw material, uses protective agent to protect beta-hydroxyl at 3-position, and utilizes mycobacteria to ferment and prepare dehydroepiandrosterone, and the method effectively improves the yield of dehydroepiandrosterone, but the mycobacteria fermentation step adopts oil fermentation conversion, the product fermentation time is long, various products are easy to produce, the processing, separation and purification are difficult, and the method is worthy of further improvement. Patent No. CN201410414313.1 discloses a method for producing dehydroepiandrosterone by microbial fermentation, 3 beta-hydroxysteroid dehydrogenase encoding gene MSMEG _5228 in mycobacterium smegmatis is knocked out, the dehydroepiandrosterone is produced by transforming phytosterol by single-aqueous phase fermentation of recombinant mycobacterium smegmatis, the transformation rate is improved, the influence of waste oil generated in microbial fermentation on environment is solved, genes are knocked out, recombinant strains are constructed, the method relates to the field of genetic engineering, the investment is large, the requirement is high, and the process is complicated.

The strain Mycobacterium sp.B-NRRL 3683 is described in U.S. Pat. No. 4755463 and is generally used only for the fermentation of phytosterols to 4-AD and ADD, and it is currently widely studied to improve the yield of 4-AD and ADD by controlling the reaction conditions.

Disclosure of Invention

In view of the technical problems, the invention aims to provide a method for preparing dehydroepiandrosterone by oil-free phytosterol conversion from growing cells, which has the advantages of high yield, high quality, low pollution and easy operation. In order to achieve the purpose, the invention adopts the technical scheme that:

a method for preparing dehydroepiandrosterone by oil-free transformation of phytosterol by growing cells comprises the following steps:

(1) and (3) protecting hydroxyl: utilizing methylal as a protective agent, protecting 3-position hydroxyl of phytosterol to obtain phytosterol etherate, and adding phosphorus pentoxide as a catalyst and diatomite as a filter aid in the reaction process;

(2) and (3) biological transformation of growing cells: carrying out slant culture and seed culture on a Mycobacterium (Mycobacterium sp.) B-NRRL 3683 strain, inoculating the strain into a transformation medium, and carrying out fermentation transformation on the phytosterol etherate to obtain a fermentation product;

(3) hydrolysis: extracting the fermentation product with ethyl acetate, heating under reduced pressure, concentrating, and hydrolyzing with hydrochloric acid to obtain hydrolysate;

(4) refining: and refining and purifying the hydrolysate to obtain a dehydroepiandrosterone product.

Preferably, the mass ratio of the methylal to the phytosterol in the step (1) is 10-20: 1.

Preferably, the method for seed culture of B-NRRL 3683 strain in the step (2) comprises the following steps:

(1) slant culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, agar 20g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, culturing at 30 ℃ for 4-5 days;

(2) first-order seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; after inoculation, shake culturing is carried out for 48h at 30 ℃ and 200 rpm;

(3) secondary seed culture: the formula of the culture medium is as follows: peptone 0.1-10g/L, yeast extract 0.1-10g/L, glucose 0.1-10g/L, disodium hydrogen phosphate 0.1-10g/L, pH 7.5-8.0, sterilizing at 121 deg.C for 30 min; inoculating the primary seed liquid to a secondary seed culture medium according to the volume ratio of 10%, and after inoculation, performing shake culture at 30 ℃ and 200rpm for 48 h.

Preferably, the transformation medium formula in the step (2) comprises the following components in percentage by mass: 1-6% of corn steep liquor, 0.1-0.6% of sodium nitrate, 0.01-0.08% of diammonium hydrogen phosphate, 0.1-0.2% of PPE, 1-10% of phytosterol etherate, 0.1-40% of hydroxypropyl cyclodextrin and pH value of 7.5-8.0.

Preferably, the phytosterol etherate is ground to 200 mesh.

Preferably, the method for transforming the growing cells in the step (2) is specifically as follows: preparing transformation medium according to the formula, sterilizing at 121 deg.C for 30min, cooling to room temperature, inoculating B-NRRL 3683 strain secondary seed liquid with mass percent of 10-20% under aseptic condition, and transforming at 28-32 deg.C, air flow of 0.5-1.0vvm, and tank pressure of 0.05-0.06 MPa.

Preferably, the mass fraction of the hydrochloric acid in the step (3) is 5%, the mixture ratio of the materials is 1-20 parts by volume of ethyl acetate and 1.5-30 parts by volume of 5% hydrochloric acid; reducing pressure and heating to-0.08 MPa, 45 deg.C, and hydrolyzing for 5 hr, adding hydrochloric acid, heating to 60 deg.C, and hydrolyzing for 1-2 hr.

Preferably, the refining method in the step (4) is specifically as follows: drying the hydrolysate at 70 ℃, dissolving by using methanol, carrying out suction filtration, carrying out reduced pressure concentration on the filtrate to a small volume, cooling to about 4 ℃, carrying out suction filtration, and drying to obtain a crude product; adding petroleum ether into the crude product, refluxing and pulping at 70 ℃ for 2-3h, cooling to 25 ℃, filtering to obtain a white-like solid, drying, adding methanol, heating to 70 ℃ for dissolution, concentrating under reduced pressure to be pasty, slowly cooling to 0-4 ℃, growing crystals for 2 hours, carrying out suction filtration, and drying.

Preferably, the material proportion used in the refining process of the step (4) is 3-6 parts by volume of methanol, 0.5-1 part by volume of petroleum ether, and 2 times of methanol addition, wherein the first use amount is 1-2 parts, the second use amount is 2-4 parts, the reduced pressure and temperature rise conditions are-0.08 MPa, 45 ℃ and 8 hours, and the drying temperature after crystal growth is 60 ℃.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention takes the phytosterol as the raw material to produce the dehydroepiandrosterone, the raw material is easy to obtain, and the production cost is reduced.

2. The method for preparing dehydroepiandrosterone by oil-free transformation of phytosterol by growing cells has the advantages of short transformation time, high product yield and high HPLC purity, less waste oil generation, less pollution and good economic benefit, and is more suitable for industrial production.

3. The activity of 3-position hydroxyl of the phytosterol is very high, and the phytosterol is easily oxidized in the conversion process to cause conversion failure.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Mycobacterium (Mycobacterium sp.) NRRLB-3683 used in the following examples was purchased from Shanghai Hainan industries, Ltd, and the phytosterol used was BETA-sitosterol, purchased from Vickers Biotech, Inc., Sichuan province.